Most intracellular proteolysis in eukaryotes requires the attachment of ubiquitin (Ub) to a substrate prior to its degradation by the 26S proteasome. Specificity in this process is determined not only by the selection of ubiquitination targets, but also by the partitioning of conjugates between two fates: (i) degradation by the 26S proteasome, and (ii) removal of Ub by Ub-protein isopeptidase(s) to regenerate the intact protein substrate. That an isopeptidase can "edit" conjugates and rescue some proteins from degradation is suggested by the principal investigator's finding that isopeptidase inhibitors selectively enhance the proteolysis of poorly ubiquitinated substrates. The principal investigator has discovered a UCH-family isopeptidase, UCH37, able to perform this editing function in vitro. UCH37 is part of the PA700 (19S) regulatory complex of the proteasome, where it is specific for removing Ub only from the distal ends of polyUb chains. Remarkably, free UCH37 is proximal-end specific and cannot cut Ub-Ub linkages at all. This dramatic specificity switch plus other findings suggest that UCH37 exists in vivo in cytosolic and nuclear complexes that have distinct functions, and that isopeptidase specificity is controlled by intersubunit interactions within the complexes. The principal investigator will investigate the molecular basis for this specificity change. The identities and functions of UCH37 complexes, and the putative editing role of proteasomal UCH37, will be studied. Specific goals are 1) to identify subunit interactions that control isopeptidase specificity in the PA700 and non-proteasomal UCH37complexes; 2) to construct Drosophila and S. pombe UCH37 mutants and to resolve their phenotypes, including loss of the editing function, from among loss of catalytic activity, structural perturbation of the 26 S proteasome, and elimination of the nuclear non-proteasomal UCH37 as possible causes; 3) to test whether displacement of the -20-residue UCH active-site-crossover loop is the critical feature that controls binding of large substrates. To do this, the principal investigator will alter the loop in UCH37 or the related Yuh1 enzyme by enlargement or specific proteolysis, or for Yuh1 (for which a crystal structure is available), he will eliminate the loop entirely by circular permutation. Steady-state and presteady-state kinetics will be used to probe active-site-crossover loop movement in Yuh1 and to test a model in which slow displacement of the loop limits to approximately1 percent the fraction of Yuh1 molecules able to bind large substrates.